Angiogenic and pleiotropic effects of VEGF165 and HGF combined gene therapy in a rat model of myocardial infarction

<div><p>Since development of plasmid gene therapy for therapeutic angiogenesis by J. Isner this approach was an attractive option for ischemic diseases affecting large cohorts of patients. However, first placebo-controlled clinical trials showed its limited efficacy questioning further advance to practice. Thus, combined methods using delivery of several angiogenic factors got into spotlight as a way to improve outcomes. This study provides experimental proof of concept for a combined approach using simultaneous delivery of VEGF165 and HGF genes to alleviate consequences of myocardial infarction (MI). However, recent studies suggested that angiogenic growth factors have pleiotropic effects that may contribute to outcome so we expanded focus of our work to investigate potential mechanisms underlying action of VEGF165, HGF and their combination in MI. Briefly, Wistar rats underwent coronary artery ligation followed by injection of plasmid bearing VEGF165 or HGF or mixture of these. Histological assessment showed decreased size of post-MI fibrosis in both—VEGF165- or HGF-treated animals yet most prominent reduction of collagen deposition was observed in VEGF165+HGF group. Combined delivery group rats were the only to show significant increase of left ventricle (LV) wall thickness. We also found dilatation index improved in HGF or VEGF165+HGF treated animals. These effects were partially supported by our findings of c-kit+ cardiac stem cell number increase in all treated animals compared to negative control. Sporadic Ki-67+ mature cardiomyocytes were found in peri-infarct area throughout study groups with comparable effects of VEGF165, HGF and their combination. Assessment of vascular density in peri-infarct area showed efficacy of both–VEGF165 and HGF while combination of growth factors showed maximum increase of CD31+ capillary density. To our surprise arteriogenic response was limited in HGF-treated animals while VEGF165 showed potent positive influence on a-SMA+ blood vessel density. The latter hinted to evaluate infiltration of monocytes as they are known to modulate arteriogenic response in myocardium. We found that monocyte infiltration was driven by VEGF165 and reduced by HGF resulting in alleviation of VEGF-stimulated monocyte taxis after combined delivery of these 2 factors. Changes of monocyte infiltration were concordant with a-SMA+ arteriole density so we tested influence of VEGF165 or HGF on endothelial cells (EC) that mediate angiogenesis and inflammatory response. In a series of <i>in vitro</i> experiments we found that VEGF165 and HGF regulate production of inflammatory chemokines by human EC. In particular MCP-1 levels changed after treatment by recombinant VEGF, HGF or their combination and were concordant with NF-κB activation and monocyte infiltration in corresponding groups <i>in vivo</i>. We also found that both–VEGF165 and HGF upregulated IL-8 production by EC while their combination showed additive type of response reaching peak values. These changes were HIF-2 dependent and siRNA-mediated knockdown of HIF-2α abolished effects of VEGF165 and HGF on IL-8 production. To conclude, our study supports combined gene therapy by VEGF165 and HGF to treat MI and highlights neglected role of pleiotropic effects of angiogenic growth factors that may define efficacy via regulation of inflammatory response and endothelial function.</p></div

Effects of human VEGF165 and HGF on IL-8 and MCP-1 production by endothelial cells <i>in vitro</i>.

<p>A—MCP-1 production by HUVEC after stimulation with VEGF165, HGF or both factors over 4–12 hrs. Student’s t-test (Mean±S.D.; n = 4): * increased MCP-1 compared vs. HSA (p<0.05); # decreased MCP-1 compared vs. HSA or VEGF (p<0.01); ## decreased MCP-1 compared vs. VEGF (p<0.03). B—Autoradiograms of membrane stained for pIκBα и IκBα after Western blotting of extracts from HUVEC stimulated by VEGF165 (25 ng/ml), HGF (25 ng/ml) or VEGF165+HGF (25 ng/ml total); HSA (25 ng/ml) served as a negative, and TNF-α (5 ng/ml) as a positive control. C—IL-8 production by HUVEC after stimulation with VEGF165, HGF or both factors over 4–12 hrs. Student’s t-test (Mean±S.D.; n = 3–4): * increased IL-8 compared vs. HSA (p<0.05); ** increased IL-8 compared vs. VEGF or HGF (p<0.001); *** increased IL-8 compared vs. VEGF or HGF (p<0.01). D—Activation of HIF and IL-8 promoter in EA.hy926 after treatment with VEGF, HGF and their combination. Data of luciferase-based reporter assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197566#sec002" target="_blank">Materials and methods</a> for detailed description) Mann-Whitney U-test (Mean±S.D.; n = 3–4): *p<0.05 vs HSA; **p<0.025 vs. VEGF or HGF.</p

Quantitative analysis of c-kit+ CSC and Ki-67+ cardiomyocyte density.

<p>(A) Representative images of sections co-stained for c-kit/troponin-I (upper panel) and Ki-67 (lower panel); (B) Statistical analysis of c-kit+ CSC and Ki-67+ cardiomyocyte counts. n = 4-5/group, data presented as Mean±S.D.; *p<0.05 vs “pC4W” negative control (Mann-Whitney’s U-test). No significant differences were found in comparison of “VEGF+HGF” vs. “VEGF” or “HGF” groups.</p

Morphometry of LV sections obtained from study groups.

<p>A—Representative images of stained sections of myocardium obtained at day 14 of experiment (fibrotic depositions stain blue); B—Results of morphometry in experimental groups treated by VEGF165, HGF or combined gene delivery; Student’s t-test (n = 4-5/group, presented as Mean±S.E.M.). LV fibrosis size graph: *p<0.05 vs “pC4W” negative control; **p<0.05 vs. “pC4W” negative control and vs. “VEGF” or “HGF” group. LV thickness graph: N.S.–not significant vs. “pC4W”; # p<0.05 vs. “pC4W”. Dilatation index graph: N.S.–not significant vs. “pC4W”; $ p<0.05 vs. “pC4W”. C—Representative images of myocardium cross-section stained for laminin (left panel) and results of cardiomyocyte length analysis in respective study groups (right graph). No significant intergroup variability was found (p>0.05).</p

Influence of VEGF165 and HGF gene delivery on monocyte infiltration of peri-infarct area.

<p>A—Photomicrographs of immunohistochemical visualization of monocytes in myocardium samples obtained at day 3 after MI and injection of pDNA; B—Results of evaluation showing changes of CD68+ cells density per mm² of tissue at days 3 and 7 of experiment (data presented as Mean±S.E.M.). Student’s t-test (n = 4/group): *significant increase of CD68+ cells density (p<0.05 vs. “pC4W” negative control); **significant decrease of CD68+ cells density (p<0.05 vs. “pC4W” at corresponding time point).</p

Influence of HIF-1/2α «knockdown» on IL-8 production by endothelial cells (in response to VEGF, HGF or their combination.

<p>EA.hy926 cells were transfected with respective siRNA and after 24 hrs were incubated with human serum albumin (HSA), VEGF165, HGF or their combination for 6 hrs. Student’s t-test (mean±S.D, n = 4): p-values are given at the graph.</p

Evaluation of angio- and arteriogenesis after gene delivery of VEGF165 and HGF.

<p>A—Representative images of peri-infacrt area in cross section of myocardium after immunofluorescent visualization of CD31 and α-SMA (upper row of images in ×200 magnification emphasizes capillary morphology, lower row in ×100 magnification shows representative examples of arteriole-sized vessels); B—Results of vascular density analysis in groups treated by VEGF165, HGF or combined gene delivery (data presented as Mean±S.E.M.). Mann-Whitney U-test (n = 4-5/group): *p<0.05 vs. “pC4W” negative control; **p<0.01 vs. “pC4W”, “HGF” or “VEGF+HGF” groups; N.S.–non-significant (p>0.05 vs. “pC4W” negative control).</p

Detection of human VEGF165 and HGF in rat myocardium explants.

<p>Primary tissue was harvested at days 3 or 7 after DNA injection (A) or homogenates of left ventricle harvested at day 3 after DNA injection (B); n = 2 animals per column, ELISA data presented as Mean±S.D.</p